Method and Apparatus for Substantially Isolating Plant Tissues

ABSTRACT

The present invention discloses and claims methods and devices for the rapid mechanical isolation of monocot plant tissues suitable for transformation or tissue culture. The invention includes mechanical devices for substantially isolating target plant tissues for use as transformable explants, and propagation of transgenic plants and plant tissues.

This application is a continuation-in-part of U.S. application Ser. No.10/911,191, filed Aug. 4, 2004, which claims the benefit of priority ofU.S. Provisional Application No. 60/493,011, filed Aug. 5, 2003, both ofwhich are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to plant propagation andmechanical methods for substantially isolating target plant tissues,such as embryos, which are suitable for genetic transformation or tissueculture.

BACKGROUND OF THE INVENTION

The preparation of tissues for plant propagation, regeneration andtransformation is time consuming and labor intensive, especially as itusually involves manual excision of transformable or culturable planttissues. For example, in corn (Zea mays), individual immature embryosare typically removed manually to provide genetically-transformableexplants. The manual excision of embryogenic tissues is laborious andrisks ergonomic injury to the worker. Moreover, when larger amounts oftransformable plant tissue are required for high-throughputtransformation and plant production, more workers must be employed andtrained to meet the increased demands. Additionally, there can besignificant variability in the quality of plant tissues obtained,depending on the skill level, care, attentiveness, and fatigue of theindividual workers. This tissue variability is problematic, as poorquality tissues negatively impact the efficiency of subsequent tissueculture, genetic transformation, and plant propagation. Thus, there is aneed in the art for methods of preparing target plant tissues that aremore rapid, reduce the overall ergonomic burden on workers, reduce theamount of workers needed to process the plant materials, and/or yieldplant tissues that are of higher quality or more consistent quality thanmanually produced tissues.

SUMMARY OF THE INVENTION

The present invention discloses methods and apparatuses to simplify,improve safety, increase reliability, reduce ergonomic injury, reducethe number of personnel required, and/or increase the speed with whichtarget plant tissues are substantially isolated for use in plant tissueculture and genetic transformation. In particular, the present inventiondiscloses and claims methods and apparatuses useful for substantiallyisolating embryos. In some embodiments, the methods and devices may beused to substantially isolate monocot embryos, such as corn embryos. Thesubstantially isolated embryos are preferably suitable for genetictransformation or tissue culture. The methods and apparatuses disclosedherein are particularly useful for high-throughput processing (i.e.,substantially isolating large numbers of target tissues and/orprocessing large quantities of seeds).

One aspect of this invention includes methods for substantiallyisolating target tissues from monocots, such as embryos, that aresuitable for genetic transformation or tissue culture. The methodcomprises (a) providing monocot seeds containing immature embryos thathave an opening in the pericarp or seed coat of the seeds; and (b)applying force to the seeds sufficient to substantially isolate theimmature embryos from the seeds. In some embodiments, immature cornembryos are substantially isolated from corn seeds. The immature embryosthus obtained are preferably suitable for genetic transformation ortissue culture.

Another aspect of this invention provides an apparatus for substantiallyisolating target plant tissues, such as embryos, suitable for genetictransformation or tissue culture. The device comprises at least oneaperture for guiding a fluid stream. In one embodiment, the fluid streamcontacts kernels on an ear of corn and causes the embryos to becomesubstantially isolated from the kernels. The substantially isolatedembryos are preferably suitable for genetic transformation or tissueculture.

In one embodiment, the apparatus comprises at least one componentselected from among (a) at least one solid surface suitable for applyingmechanical positive pressure to the exterior of a seed; (b) at least oneaperture for guiding a fluid flow; (c) at least one aperture forapplying negative fluid pressure; and any combinations thereof. Thecomponent may be used to direct a physical force on the seed sufficientto substantially isolate a target tissue, such as an embryo.Accordingly, the apparatus may be used to substantially isolate cornembryos suitable for genetic transformation or tissue culture from anear of corn. In some embodiments, the aperture for guiding a fluid flowdirects the fluid flow to contact corn seeds or kernels on an ear ofcorn. In some embodiments, the aperture for applying negative fluidpressure directs the negative fluid pressure to contact corn seeds orkernels on an ear of corn. The target tissues substantially isolated bysuch an apparatus are preferably suitable for tissue culture or genetictransformation.

The invention further provides transgenic plants, plant tissues, andseeds. Transgenic plant and plant tissues may be produced by (a)substantially isolating a target tissue using the methods and/orapparatuses described herein, (b) introducing a heterologous nucleicacid molecule into the target tissue to produce a transformed explant,and (c) culturing the transformed explant under suitable growthconditions to produce a transgenic plant tissue or plant. Any method oftransformation is suitable and known to those of skill in the art.Additionally, suitable culture and regeneration conditions are known androutine. The transgenic plant, plant tissue, or seed is preferably amonocot, such as corn. The invention also includes all progeny plants,plant tissues, and seeds that are produced from the transgenic planttissue or plant.

Other embodiments of the invention are disclosed in the followingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts one embodiment of an apparatus of the present inventionthat uses positive mechanical pressure for substantially isolatingembryos, as described in Example 4.

FIG. 2 depicts one embodiment of an apparatus of the present inventionthat uses fluid jet positive pressure to dislodge embryos from seeds bya method of the invention, as described in Example 7. Legend: (A) robotwith motion in X, Y and Z dimensions, (B) motor to rotate corn ear, (C)grasper, (D) handle to hold corn ear, (E), baffle to prevent materialfrom splattering upwards, (F) flange to prevent material splatteringupwards, (G) aperture for guiding fluid, (H) transparent tube, (I) cornear, (J) shaking screen, (K) cheesecloth or other porous material, and(L) waste container.

FIG. 3 depicts one embodiment of a mounting mechanism using a magnetic“handle” by which a corn ear can be secured to a robot arm, as describedin Example 7.

FIG. 4 depicts one embodiment of a nozzle useful in methods of theinvention, as described in Example 7. This nozzle generates asubstantially uniform, flat sheet-like jet of fluid.

FIG. 5 depicts one embodiment of an apparatus useful in methods of theinvention, as described in Example 13. This device includes a nozzle forgenerating a substantially flat fluid jet and an optional suction head.FIG. 5 (top) depicts a cross-sectional view of one embodiment of such adevice, showing how the nozzle, optional suction head, and corn ear maybe positioned relative to each other. FIG. 5 (bottom) schematicallydepicts a corn ear positioned in the device. Legend: (A) base, (B)holder, (C) nozzle, (D) suction head, (E) corn ear, and (F) aperture forguiding fluid flow.

FIG. 6 depicts one embodiment of a component useful for applyingnegative fluid pressure useful in methods of the invention, as describedin detail in Example 13. Legend: (A) one or more apertures for guidingfluid flow.

FIG. 7 depicts FIG. 7A through 7C depict different views of anembodiment of a device that uses a combination of forces and is usefulin methods of the invention, as described in detail in Example 13. Thisdevice includes a head with a leading edge capable of applying apredefined amount of mechanical pressure to the base of kernels thatpreviously have had the pericarp opened or truncated and a component forapplying negative fluid pressure. This device can further include ameans for dispensing fluid or for guiding fluid flow.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs, when taken in context of thepresent specification. Where there are inconsistencies between the textof the specification and the material incorporated by reference, thedefinitions and meanings provided in the present specification areintended. The nomenclature used herein and the manufacture or laboratoryprocedures described below are well known and commonly employed by thoseof skill in the art.

The phrases “substantially isolated” or “extracted” refer to theprocessing of a target tissue (e.g., an embryo or other tissue explant)that resides in or forms part of a larger tissue complex (e.g., a seed)such that the target tissue is physically separated from at least halfof the larger complex. In some embodiments, a substantially isolatedtarget tissue may be physically separated from at least about 50%, 60%,70%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the larger complex, or anyfraction thereof. In other embodiments, the target tissue is physicallyseparated from more than about 80% to about 100%, about 90% to about100%, or about 99% to about 100% of the larger complex, or any fractionin between. In some embodiments, the target tissue may be physicallyseparated from about 100% of the larger complex.

While a substantially isolated target tissue is physically separatedfrom some percentage of the larger complex, it does not necessarily haveto be purified from that complex. In other words, the substantiallyisolated target tissue may remain in a batch with the larger tissuecomplex, so long as the target tissue is physically separated from thecomplex (as described above). In some embodiments, however, it may bedesirable to remove some or all of the separated complex from thesubstantially isolated target tissue. All such embodiments are withinthe scope of the present invention.

The phrase “target plant tissue” refers to a portion of a plant tissueor seed that one seeks to substantially isolate. In the presentinvention, target plant tissue refers to any portions of a plant orplant seed that can be substantially isolated and used for genetictransformation or tissue culture. In some embodiments, the target planttissue is an embryo, in particular, an immature embryo from a monocotsuch as corn.

The phrase “suitable for genetic transformation” and “suitable fortissue culture” refer to plant tissues that are competent fortransformation or growth in under suitable plant culture conditions,respectively. One of skill in the art can readily determine if aparticular target tissue is suitable for genetic transformation ortissue culture by using routine experimentation. For example, a samplefrom a batch of substantially isolated target tissues may be cultured ona suitable plant media (also known to those of skill in the art) todetermine if the tissues are capable of growth and regeneration.Similarly, samples of substantially isolated target tissues can besubject to transformation and screened for the presence of aheterologous nucleic acid molecule. Such techniques are routine and canrapidly identify which tissues are competent for transformation ortissue culture and which, if any, are not.

Where a term is provided in the singular, the inventors also contemplateaspects of the invention described by the plural of that term.

Methods for Substantially Isolating Target Plant Tissues

The present invention provides methods of substantially isolating targetplant tissues suitable for genetic transformation or tissue culture,comprising (a) providing seeds containing an opening in the pericarp orseed coat of the seeds; and (b) applying force to the seeds sufficientto substantially isolate a target plant tissue from the seeds. In someembodiments, the target plant tissue is an embryo. The embryos arepreferably monocot embryos, such as from corn. In some embodiments, thesubstantially isolated target tissue may be isolated in whole are inpart. For example, a batch of substantially isolated immature embryosmay include intact embryos, partial embryos, or mixtures thereof.Preferably, the intact and/or partial tissues are suitable for genetictransformation, tissue propagation, plant regeneration and other tissueculture applications.

Suitable procedures for plant tissue culture and regeneration are wellknown in the art. See, for example, U.S. Pat. No. 5,550,318 to Adams etal., U.S. Pat. No. 5,780,708 to Lundquist et al., United States PatentApplication Publication Number 2004/0210958 to Duncan et al., UnitedStates Patent Application Publication Number 2004/0016030 to Lowe etal., and United States Patent Application Publication Number2004/0244075 to Cai et al., which disclose transformation methods usefulwith corn, and United States Patent Application Publication Number2003/0024014 to Cheng et al., which disclose transformation methodsuseful with wheat, all of which are incorporated by reference in theirentirety herein. These tissue culture applications can include at leastone process selected from transformation, callus formation, directembryogenesis, formation of differentiated plant tissue, formation of atleast one mature plant, formation of at least one fertile mature plant,and combinations of these processes. The plants regenerated from theextracted immature embryos may be regenerated, for example, throughdifferentiation of dedifferentiated tissue (calli) or by directembryogenesis of the extracted immature embryos. Regenerated plants canpreferably be grown to maturity to provide mature plants, and morepreferably fertile mature plants. The extracted immature embryos andextracted non-embryo tissues may also be used for other purposes, suchas, but not limited to, genetic or biochemical analysis.

The methods and apparatuses of the present invention can be applied toany monocot plants of interest. Preferred monocots include, but are notlimited to, members of the family Poaceae, including grasses such asturf grasses and grain crops such as corn (maize), wheat, and rice.Particularly preferred monocots include Zea species, including corn (Zeamays), which has multiple kernels (seeds) typically held in rows on acorn ear.

In general, the monocot seeds from which the target tissues aresubstantially isolated are provided in any suitable manner. For example,seeds may be attached to the ear or head on which the seeds grow; insome embodiments the monocot seeds may be removed from the ear or headprior to substantially purifying the target tissue.

In some embodiments, an opening in the pericarp or seed coat of themonocot seeds is provided. This may be accomplished by any suitabletechnique, such as, but not limited to, making a hole, puncture, orincision with a needle, awl, blade, or other suitable implement. In someapplications of the method, no pericarp tissue need be removed; in otherembodiments, the opening of the pericarp may include removal of at leastpart of the pericarp and possibly of some non-embryo tissue (e.g.,endosperm). Preferably, the opening is sufficient to substantiallyseparate the embryo from the seed. In some embodiments it may benecessary only to weaken the pericarp sufficiently (for example, byabrasion, or by other physical, chemical, or enzymatic treatment) sothat application of force to the seed results to substantial isolationof the target tissue, such as the embryo.

The method includes the step of applying force to the seeds sufficientto substantially isolate the target tissue, such as an immature embryo,from the seeds, wherein the substantially isolated target tissue issuitable for genetic transformation and tissue culture. Force may beapplied to multiple seeds consecutively or simultaneously. The appliedforce can be continuous or non-continuous (for example, pulsed orwave-like force), and is generally mechanically applied, that is to say,the force is obtained through the use of a device or machine rather byhuman hand. The amount of force applied is preferably sufficient toovercome the adhesion of the target (e.g., embryo) and non-target (e.g.,non-embryo tissue such as endosperm) from each other, thus allowingseparation of the target and non-target tissues. Any suitable force orforces may be employed for removal of the target tissue from its seed,and multiple forces may be used in combination, sequentially orsimultaneously. Suitable forces include, but are not limited to, fluidjet positive pressure, liquid jet positive pressure, mechanical positivepressure, negative pressure, centrifugal force, linear acceleration,linear deceleration, fluid shear, fluid turbulent flow, and fluidlaminar flow. Fluid forces can be exerted by any fluid, gases or liquidsor combinations of both.

The method can further include the step of separating the substantiallyisolated target tissue, such as immature embryos, from associatednon-embryo tissue such as endosperm, glumes, and seed coat or pericarptissues. Separation may be accomplished by one or more suitabletechniques, including, but not limited to, separation by size exclusion(for example, by filtration in one or more filtering steps), separationbased on hydrophobicity, hydrophilicity, lipophilicity, or otherattractive forces, and separation by mass or density differentials (forexample, separation by centrifugation, settling, and decanting). Theseparation step or steps can be optional, for example, where noadditional isolation of intact or partial embryos is necessary for theiruse in tissue culture.

The method of the invention is particularly suitable to applicationswhere a large number of target tissues must be provided, for example, inhigh-throughput processes or screening, or in batch processing forgenetic transformation or tissue culture. Automation of the method ispossible, for example, by employing robotic or mechanical handling ofthe corn ears or seeds, opening of the pericarp, application of force tothe seed, or the optional separation steps. Such automation may useoptical or mechanical sensors to aid in positioning the corn ears orseeds relative to the applied force or forces, or in the separationsteps. In one preferred embodiment, the method provides substantiallyisolated embryos at a rate of between about 250 to 100,000 or moreembryos per employee-day; or between about 250 to about 100,000, orabout 250 to about 50,000, or about 250 to about 20,000, or about 250 toabout 10,000, or about 250 to about 5000, or about 250 to about 3000, orabout 250 to about 1000 embryos per employee-day; or between about 800to about 100,000, or about 800 to about 50,000, or about 800 to about20,000, or about 800 to about 10,000, or about 800 to about 5000, orabout 800 to about 3000, or about 800 to about 1000 embryos peremployee-day; or between about 2500 and about 100,000, or about 2500 toabout 50,000, or about 2500 to about 20,000, or about 2500 to about10,000, or about 2500 to about 5000, or about 2500 to about 3000 embryosper employee-day; or between about 5000 and about 100,000, or about 5000to about 50,000, or about 5000 to about 20,000, or about 5000 to about10,000 embryos per employee-day, or any fraction or whole number inbetween any of the aforementioned ranges. As a reference, anemployee-day is equivalent to one day's labor of one employee of averageskill in the art. While average employee output can vary, the followingis given as a guideline for purposes of comparing the present inventionwith the current average employee output. To manage the ergonomicburden, it is currently suggested that workers excise about one ear ofcorn per a day (approximately 200 to 300 embryos) about twice per week.Thus, an average employee following such recommendations can produce upto about 600 embryos per week. It is possible that an average employeecould produce up to about 500-800 excised embryos in one day. However,maintaining such an output over the course of several days or even weeksis not recommended due to the increased ergonomic burdens and qualityconcerns. As noted above, the present invention overcomes thesesignificant output limitations.

Apparatuses for Substantially Isolating Target Plant Tissues

The present invention also provides apparatuses for substantiallyisolating target tissues, such as corn embryos, that are suitable forgenetic transformation or tissue culture. In an embodiment forseparating corn embryos, such an apparatus comprises at least oneaperture for guiding a fluid stream, wherein the fluid stream contactskernels on the corn ear and substantially isolates embryos from thekernels. Generally, it is preferred that the fluid stream contact asmany of the kernels in a given period of time as is convenient, so as tomore rapidly isolate embryos. The at least one aperture can include asingle aperture or multiple apertures (for example, single or multiplenozzles, which can include flat, round, oval, fan-shaped or otherpatterned nozzles, and adjustable, moving, or stationary nozzles), andcan generate a fluid flow of any suitable type and medium. Fluids may begases (such as air, nitrogen, or gas mixtures), liquids (such as water,physiological saline, or various culture media), or combinations.Suitable fluid flows include, but are not limited to, fluid jets (suchas single or multiple columnar jets; flat, cone-shaped, or fan-shapedjets or sprays; and sheet-like jets), laminar fluid flow, and turbulentfluid flow. Suitable fluid flows can result in a variety of forces toremove the embryo from its kernel, including positive pressure ornegative pressure or both; such forces can be uniform or non-uniform,continuous or non-continuous (such as a pulsed or wave-like force), orin any combination thereof.

The apparatus of the invention may further include a means for movingthe target tissue being substantially purified and the fluid stream,relative to each other. For example, either the ear of corn containingseeds or the fluid stream, or both, may be moved. Various embodiments ofthe apparatus can be used with single or multiple, intact or partialears of corn. For example, the corn ear or ears can be secured to aholder or grasper, which is moved relative to the fluid stream. In otherembodiments, however, the corn ear or ears need not be individuallysecured to a holder but can be freely movable so as to allow multiplekernels to be contacted by the force used to remove the embryos from thekernels. The means for moving at least one corn ear relative to thefluid stream can rotate the at least one corn ear and the at least oneaperture relative to each other, or can move the fluid stream along thelongitudinal axis of the at least one corn ear, or can provide anysuitable three-dimensional movement of the at least one corn ear and theat least one aperture relative to each other, such as a combination ofrotation and longitudinal motion.

The apparatus of the invention can further include at least oneseparator for separating target tissues from non-target tissues. Forexample, embryos may be separated from non-embryo tissues, wherein theseparated embryos comprise at least some corn embryos suitable forgenetic transformation or tissue culture. Separators can work by anysuitable mechanism, including, but not limited to, separation by sizeexclusion (for example, using a mesh, screen, perforated surface, orother device capable of excluding objects of a certain size), separationbased on hydrophobicity or other attractive forces (for example, using amaterial, solid or fluid, that can attract or repel the embryos), andseparation by mass or density differentials (for example, using acentrifuge, or using solutions for differential settling). In certainembodiments, the at least one separator can be optional, for example,where no additional isolation of intact or partial embryos is necessaryfor their use in genetic transformation or tissue culture.

The substantially isolated (and optionally separated) immature embryosinclude at least some embryos, such as immature intact or partialembryos, suitable for tissue culture applications, transformation,callus formation, direct embryogenesis, formation of differentiatedplant tissue, formation of at least one mature plant, formation of atleast one fertile mature plant, and combinations of these processes, asdescribed above. The substantially isolated immature embryos andnon-embryo tissues may also be used for other purposes, such as, but notlimited to, genetic or biochemical analysis.

The present invention further provides an apparatus for mechanicallysubstantially isolating multiple corn embryos suitable for genetictransformation or tissue culture from at least one immature corn ear,including at least one component selected from (a) at least one solidsurface for applying mechanical positive pressure to the exterior ofkernels on the at least one immature corn ear; (b) at least one aperturefor guiding a fluid flow, wherein the fluid flow contacts kernels on theat least one immature corn ear; and (c) at least one aperture forapplying negative fluid pressure, wherein the negative fluid pressurecontacts kernels on the at least one immature corn ear; and wherein theat least one component applies force to the kernels sufficient tosubstantially isolate embryos from the kernels, the substantiallyisolated embryos including multiple immature embryos suitable forgenetic transformation or tissue culture. A suitable apparatus appliesone or more forces sufficient to substantially isolate the immatureembryos from the seeds, wherein the substantially isolated immatureembryos include embryos suitable for genetic transformation or tissueculture. The one or more forces may be applied to multiple seedsconsecutively or simultaneously, in a continuous or non-continuousmanner, and is generally applied mechanically and not manually. Multipleforces may be used in combination, sequentially, or simultaneously.Suitable forces include, but are not limited to, fluid jet positivepressure, liquid jet positive pressure, mechanical positive pressure,negative pressure, centrifugal force, linear acceleration, lineardeceleration, fluid shear, fluid turbulent flow, and fluid laminar flow.Fluid forces can be exerted by any fluid, gases or liquids orcombinations of both.

Combination apparatuses of the invention can optionally include a meansfor moving the at least one corn ear relative to the source or sourcesof force (that is to say, the solid surface for applying mechanicalpositive pressure, the aperture for guiding a fluid flow, or theaperture for applying negative fluid pressure). Preferably the ear orears is moved relative to the source of force so that the force orforces contact as many of the kernels in a given period of time as isconvenient, so as to more rapidly isolate embryos.

Combination apparatuses of the invention can further include at leastone means for further separation of the substantially isolated immatureembryos suitable for genetic transformation or tissue culture, whereinthe separated embryos comprise at least some corn embryos suitable forgenetic transformation or tissue culture. Separators can work by anysuitable mechanism, including, but not limited to, separation by sizeexclusion, separation based on attractive forces, and separation by massor density differentials.

Transformed Plants and Methods of their Production

The present invention also provides a transformed monocot plant,produced by the steps including (a) providing at least one transformabletarget tissue using the methods or apparatuses described herein; (b)introducing a heterologous nucleic acid molecule into the transformabletarget tissue to produce a transformed explant; and (c) growing atransformed monocot plant from the transformed explant. Preferredmonocots of the invention are transformed members of the family Poaceae,including grasses such as turf grasses and grain crops such as corn(maize), wheat, and rice.

Particularly preferred monocot plants include transformed Zea species,such as Zea mays. Transformed corn preferably contains at least oneheterologous nucleic acid molecule capable of conferring a desired traitto the transformed corn, such as herbicide resistance, pest resistance,cold germination tolerance, water deficit tolerance, increasedproductivity, increased yield, and the like. Practical transformationmethods and materials for making transgenic monocot plants of thisinvention (for example, various media and recipient target cells,transformation of immature embryos, and subsequent regeneration offertile transgenic plants) are disclosed, for example, in U.S. Pat. No.6,194,636 to McElroy et al., U.S. Pat. No. 6,232,526 to McElroy et al.,United States Patent Application Publication Number 2004/0216189 toHoumard et al., United States Patent Application Publication Number2004/0244075 to Cai et al., which disclose methods useful with corn, andUnited States Patent Application Publication Number 2003/0024014 toCheng et al., which discloses methods useful with wheat, all of whichare incorporated by reference herein. Single or multiple heterologousnucleic acid molecules may be used for transforming the monocot plantsof the invention; for example, constructs for coordinated decrease andincrease of gene expression are disclosed in United States PatentApplication Publication Number 2004/0126845 to Van Eenennaam et al.,which is incorporated by reference herein.

The seeds of resulting transgenic, fertile plants of the invention canbe harvested and used to grow progeny generations, including hybridgenerations, of transformed plants that include the heterologous nucleicacid molecule in their genome. Thus, the present invention includes bothprimary transformed plants (“R0” plants, produced by transformingembryos provided by a method of invention) and their progeny carryingthe heterologous nucleic acid molecule. Such progeny transgenic plantscan be prepared by crossing a transformed monocot plant of the inventionhaving the heterologous nucleic acid molecule with a second plantlacking the construct. Also, a transformed monocot plant of theinvention can be crossed with a plant line having other heterologousnucleic acid molecules that confers another trait to produce progenyplants having heterologous nucleic acid molecules that confer multipletraits.

EXAMPLES Example 1: Method to Extrude Multiple Corn Embryos

This example shows a method using mechanical positive pressure from anextruder device to produce embryos suitable for tissue culture orgenetic transformation.

The tops of kernels were sterily removed from an immature ear of corn(Zea mays) with a common vegetable peeler. The peeler was pushed fromthe basal end of the corn ear to the apical end using a slight sawingmotion to obtain a quick, sharp truncation of the kernels. While in thisembodiment the individual kernels are truncated to expose the interiortissues, in other embodiments, it may be necessary only to ensure thatan opening (such as a puncture or incision or abrasion) is made in thepericarp without actual removal of pericarp material. Where intactembryos are desired (for example, intact embryos for transformation),the size of any opening is preferably sufficient to allow removal of theembryo without damaging it. Opening of the pericarp can be accomplishedby using any suitable device, including, but not limited to, blades andabrasive materials. For example, a vegetable peeler is designed to berelatively safe and fast to use; it has a regulated cutting depth andalso requires less skill to use than a scalpel. Other tools with similarfunctions can be employed. The devices for opening the pericarp arepreferably sterilizable, for example, by autoclaving or heating or bychemical sterilization. These pericarp treatment processes can beautomated; for example, a blade or blades or abrader can be motorized.

A sterile extruder (in this case, a 4 millimeter diameter rod) waspushed against the base of the truncated kernels. Other suitableextruding devices may be employed. Preferably, such devices should havea size and shape capable of applying a relatively localized force to thebase of the truncated kernels to eject the embryos and endosperms.Preferably, the force applied is of sufficient magnitude and is appliedin a suitable direction such that the advancing extruder does not “rideup” over the forward kernels. The trailing edge of the extruderpreferably also provides a surface on which the ejected embryos andendosperms accumulate; for example, a flat piece of stainless steel witha rounded front edge could be used. In this example, the embryos weregently squeezed out from the pericarp, followed by the endosperms. Theextruded embryos and endosperms came to rest on the top of the advancingextruder rod, and were not crushed during the process.

The mixture of embryos and endosperms was washed with an aqueous fluidmedium (water, liquid medium, or saline) onto a sterile mesh havingdiamond-shaped openings (about 2×3 millimeters). The endosperms wereobserved to be largely retained, and the smaller embryos and somesmaller endosperm debris were washed through the screen into acollecting receptacle. The collected embryos were washed twice to removesmall debris.

The washed embryos were further purified by a flotation process. In thefirst step of the flotation process, the aqueous fluid medium wasthoroughly withdrawn from the collecting receptacle, which was allowedto dry briefly (for example, about a minute), such that remainingaqueous medium withdrew from the waxy surface of the embryos, exposingthem directly to the air. New aqueous medium was added, and the majorityof the embryos floated because their waxy surface was not rewetted bythe fluid. Non-embryo tissues such as endosperm debris remainedsubmerged in the medium, and a clear separation of embryos andnon-embryo tissues was obtained. The flotation of the extruded embryoscould be improved by more rapid, complete, or reproducible withdrawal ofthe aqueous medium, such as through the use of aspiration, or bycapillary action (e.g., of a sterile absorbent placed in the collectingreceptacle to absorb the fluid away from the extruded embryos).

A yield of approximately 100 embryos was isolated in this preliminaryexperiment, wherein only a portion of the embryo-endosperm material fromthe entire ear was processed. These results demonstrate that methods ofthe present invention are practical and convenient for harvesting largenumbers of immature embryos from corn cobs.

The embryos isolated by a method of the invention may then be used intissue culture procedures, for example, regeneration methods to generatetransgenic corn plants. Transfer of the isolated embryos to culturemedium was easily done by placing forceps, with the tips closedtogether, underneath the floating embryos, lifting them free of theliquid with the forceps and placing them on culture medium. Anothertechnique could be to pick the isolated embryos up with an instrumentthat has a hydrophobic surface. An additional technique would be totransfer embryos by hydrophobicity, for example, transferring them tothe medium surface by a small puff of air or sudden mechanical movement,such that their kinetic energy exceeds the hydrophobic force that holdsthem to the instrument.

Example 2: Visual Confirmation of Embryo Size

This example describes an improvement to one embodiment of the method ofthe present invention, as described in Example 1. Using the approachdescribed in Example 1, immature corn embryos need to be as close to thetruncated part of the kernel in order to be ejected in the greatestnumbers. Variation in immature corn embryo size is an importantconsideration in gauging the amount of kernel top to remove. Embryostend to be largest in the mid-section of the ear, with somewhat smallerembryos towards the ends. Smaller embryos, e.g., smaller than about 1.5millimeters in length are more difficult to remove unless they are closeto the truncation.

One way to ensure that enough of the kernel has been decapitated aboveembryos of varying sizes is to observe the cob during the decapitationprocess under low magnification. For example, low magnification goggles(Donegan Opti-VISOR headband binocular magnifier equipped with a No. 7lens, which provides a 2.75× magnification) were used to aid visualconfirmation of embryo size and suitable truncation of the pericarp. Ifthe first cut did not remove enough of the kernel apex, a second cutcould be made. Other low magnification devices, using the same orsimilar magnifications could be used. For example, available lenses forthe Opti-VISOR provide magnification ranging from 1.5 to 3.5×.

Example 3: Extrusion of Embryos and Endosperms

This example describes an improvement to one embodiment of the method ofthe present invention, as described in Example 1. Powered devices may beused to assist in the extrusion of embryos and endosperm. For example, apower chisel such as a WeCheer 320 power chisel, fitted with a roundedextruder device, can be used to reduce the force a person needs to exertto eject the embryos and endosperms. Other powered devices are availableand can be similarly used. Preferably, the “chisel” portion of such atool (or any part of the tool that might come into contact with theembryos) can be conveniently sterilized, for example, by insertion intoa bead sterilizer.

In one experiment, the blade of a stainless steel weighing spatula wasbent back on itself to provide an extruder device having a roundedleading edge. After insertion into a WeCheer 320 power chisel, a portionabout 10 centimeters long extended out from the power chisel's chuck.This assembly was used to eject the embryos and endosperms fromindividual rows of decapitated kernels. As the extruder device (modifiedspatula) moved down a row of kernels, a slight tendency for the spatulato slide off center to the left or right was observed; however, thistendency could be corrected by including a small keel-like extension ofthe spatula on each outer edge.

Example 4: Mechanized Embryo Extrusion

This example describes an improvement to one embodiment of the method ofthe present invention, as described in Example 1. Mechanization of theembryo extrusion process can be achieved by use of a suitable device,such as, but not limited to, the device described herein andschematically diagrammed in FIG. 1. This device includes two motors. Thefirst motor D is a stepper motor that can rotate the corn ear so thatnew rows of kernels are exposed to the two extrusion rods G, which applyforce to squeeze the embryos and endosperms out of their pericarps.

Rods G are conveniently located on opposite sides of the ear in order tobalance the pressure applied to the ear relative to the ear'slongitudinal axis. However a single rod can be used, or more than tworods; where multiple rods are used, it is preferable that they arepositioned so as to evenly distribute the resulting mechanical pressurearound the ear. The rod need not be a straight rod; in one embodiment ofthe device, a flexible “collar” encircling the circumference of the earis used instead of a rigid rod. In another embodiment, multiple shortrods or rollers are arranged in a flexible, circular configuration thatcan be slid along the ear's longitudinal axis, applying mechanicalpressure to many or all rows of kernels simultaneously.

The second motor is connected to the pinion gear E connecting to a rackF so that up and down linear motion of the ear occurs. The base of theear is held firmly to a handle B by means of a screw extending from thehandle down into the base of the ear. The narrowed middle portion of thehandle is square so that it will not rotate unless the holder C to whichit is attached is rotated by the stepper motor D.

Before insertion into the machine, the tops of the kernels aredecapitated as in Example 1 so that the embryos and endosperms can besqueezed out. To start the process, the ear is lowered until the tworods G are near the base of the ear just below the handle B. Then therods are pressed against both sides of the ear and the rack and pinionassembly draws the ear upward. As this happens, the embryos andendosperms are removed from a couple of rows, fall downward into thecollection dish H resting on the base I, and collect in a pile J. Whenthe rods approach the apical end of the cob, the cob is withdrawn upwardto its original starting position and rotated slightly by the steppermotor until new rows of kernels come into position.

Various degrees of automation of this machine are possible, includingsensors to automatically adjust the vertical starting and finishingpositions as well as the rotary start and finish positions. A rack andpinion is not the only method by which linear motion can be obtained.Pneumatics or hydraulics may be preferred for some applications. Rods Gcan be automatically opened by a suitable mechanism. When a new ear isloaded, it may be preferable to raise the ear to a position high enoughto clear the rods.

Example 5: Hydrophobic Separation of Embryos

This example describes an improvement to one embodiment of the method ofthe present invention, as described in Example 1. In separationapplications the material of interest frequently appears at theinterface of dissimilar phases (for example, between aqueous andlipophilic solvents). Removing the material of interest from such aninterface can pose problems, and has in the past been a manual processinvolving close contact with the extractant and the material to beextracted. Often the only way to successfully separate out a componentis to use a material of the same polarity orhydrophobicity/hydrophilicity. In the case of immature corn embryosextruded by a method of the invention, the embryos are found at theaqueous/air interface. The corn embryos' surface is waxy, i.e.,lipophilic or hydrophobic, and when an embryo cuticle is contacted witha substance of similar hydrophobicity, the embryo will tend to stick tothe hydrophobic surface. The embryo's hydrophobicity reduces the surfacetension of the water around it, which helps the embryo to “float” at thesurface of the aqueous/air interface.

One approach that takes advantage of these physical characteristicswould be to touch the floating embryos with a hydrophobic material (suchas hydrophobic filter paper, e.g., Whatman No. 1 PS paper, which is awater-repellant phase separating paper impregnated with silicone; see,for example,www.whatman.com/repository/documents/s3/tech_appli_010.html). In oneexample, a piece of sterile hydrophobic filter paper can be lowered ontoan entire container of floating embryos and pick them all up at once. Inanother example, a small piece of the hydrophobic paper can be used tosuccessively pick up a number of embryos and transfer them to the nextcontainer. In a third example, either a small piece of the hydrophobicpaper or a hydrophobic pipette tip would be used to contact and pick upindividual embryos and then dispense them with a puff of air from thepipettor. Ordinary pipette tips could also be modified for such use byinserting a pipette tip into a short length of hydrophobic tubing (forexample, silicone tubing); the embryo could then be picked up byhydrophobic attraction to the distal end of the hydrophobic tubing, andthen released by dispensing a puff of air from the pipette. Reducedsurface tension around the hydrophobic embryos helps them float on anaqueous surface, and the floating embryos could also be transported bymoving them on the aqueous surface (for example, by an air jet directedat the embryos). Picking up and dispensing of embryos can be automatedusing modifications of existing devices, such as machines designed forcolony picking or for retrieving protein spots on stained 2-D proteingels.

Example 6: Further Methods of Ejecting or Extruding Embryos

The method of the present invention encompasses the use of various typesof force, or combination of forces, for separating the embryo from itsseed. This example describes further embodiments. In one basic method asdescribed in Example 1, mechanical positive pressure is applied to thebase of a truncated seed (such as a corn kernel) to eject the embryo outthrough the truncated top of the seed.

In another embodiment, centrifugal force can be used to eject theembryo. For example, a corn ear (the kernels of which have previouslybeen truncated) could be spun about its longitudinal axis at a speedsufficient to eject the embryos and/or endosperms in a radialtrajectory. Spinning could be achieved by any suitable technique, suchas, but not limited to, contacting the apical end of a corn ear with afreely rotating cone, wherein the rotation of the ear is kept within alimited longitudinal range, for example, by attaching the basal end ofthe ear to a handle which is then inserted in a holder within which itcan rotate. In one exemplary embodiment using centrifugal force, about athird of the top of each kernel on a corn ear was removed with ascalpel, and the ear rolled on a surface to loosen the embryo andendosperm within the kernels. The ear was snapped into two pieces, eachabout 750 millimeters in length. Each piece was placed in a250-millilter centrifuge bottle with about 100 milliliters of water.These were centrifuged 15 minutes at 5000 rpm to eject the embryos.Examination of the ears after centrifugation showed that, in someportions of the ear, all the embryos had been removed by thecentrifugation, whereas in other areas, few or no embryos were removed.The ejected material was centrifuged and the supernatant removed toleave a slurry, which contained intact embryos (estimated to includeabout 20 percent of the total number of embryos). In another example, animmature ear of corn is harvested (typically between about 10 to about14 days post-pollination). The ear is disinfested, and under sterileconditions the top of each kernel is cut off. The ear is mounted on adrill bit on an electric drill (or a similar device) and the ear issurrounded by a large sterile collection vessel (e.g., a large glassbeaker). The ear is spun at a rotation sufficient to eject the immatureembryos, and the ejected tissues are collected from the sterilecontainer. Immature embryos are collected, for example, by manualcollection, or by rinsing the container with sterile tissue culturemedium and recovering an enriched fraction containing the embryos (e.g.,by sieving, by the use of a liquid density gradient, or by other methodsto separate embryos from non-embryo tissues as described elsewhere inthis disclosure). The immature embryos (or callus derived from theimmature embryos) can be used subsequently for transformation. Improvedresults using these and other centrifugation methods can be obtained bydetermining preferred centrifugation times and speeds by routinetesting.

Another embodiment employs bulk maceration of kernels. An immature earof corn is harvested (typically between about 10 to about 14 dayspost-pollination). The ear is disinfested. The pericarp can be openedunder sterile conditions or the kernels can be left intact. The kernelsare removed from the cob by any suitable procedure, including, but notlimited to, using a scalpel or other bladed tool. The kernels, onceseparated from the cob, are placed in tissue culture medium. Thekernel-medium mixture can be subjected to further tissue disruptionusing a suitable cutting device, such as, but not limited to, a blender.Immature embryos are collected, for example, by manual collection, or byrinsing the container with sterile tissue culture medium and recoveringan enriched fraction containing the embryos (e.g., by sieving, by theuse of a liquid density gradient, or by other methods to separateembryos from non-embryo tissues as described elsewhere in thisdisclosure). Immature embryos (or callus derived from the immatureembryos) can be used subsequently for transformation.

In a further embodiment, fluid jets (of gases or liquids or combinationsthereof) could be used to dislodge embryos. One example of this approachis to automatically rotate a corn ear in a stepwise or continuous(helical) manner past a stationary jet, collecting the ejected materialcontaining the embryos and further isolating the embryos if necessary,for example, by size separation on a mesh or screen or the like. Wherethe corn ear is vertically orientated (with respect to its longitudinalaxis), it may be preferred to rotate the ear in an upward helicaldirection, or otherwise move the ear relative to the jet so thatextracted embryos tend to wash downward.

In yet another embodiment, linear deceleration or linear accelerationcould be employed to dislodge or eject the embryos. For example, a cornear could be administered a shock parallel to the ear's longitudinalaxis and of sufficient force to eject the embryos and endosperms. A cornear could be enclosed in a suitable sterile, high impact-resistantholder, which could be subjected to sudden acceleration or deceleration,for example, by a sharp impact (e.g., as from a mallet).

Another improvement to the method would be to facilitate ejection orextrusion of the embryo from the truncated seed. For example, embryoscould be loosened or dislodged within their native position within theseed by applying a force to the tops of intact seeds (e.g., by applyinga roller or other means of applying pressure to the tops of rows of cornkernels in an intact ear or rolling or pressing the ears themselves on asurface prior to decapitating the tops of the kernels). Embryos may alsobe loosened within the seed by application of vibration, for example, byultrasound. Another approach would be to remove additional non-embryotissue, such as additional lateral wall (pericarp) material, beforeembryo ejection or extrusion. For example, a V-shaped knife or otherinstrument could be used to remove some of the lateral walls of cornkernels in rows in the ear.

Example 7: Automated Embryo Isolation Using Fluid Jet Positive Pressure

This example describes a further embodiment of the present invention. Inthis example, an automated device uses fluid jet positive pressure todislodge embryos from seeds. With reference to FIG. 2, a robotic grasperC (preferably capable of motion in three dimensions by means of robot Aand motor B, or by an equivalent means) picks up a corn ear I (by ahandle D having a baffle E) in a defined position for a rack on therobot deck. The robot inserts the corn ear into tube H (optionally madeof transparent material for ease of visual observation) at a startingposition below flange F. Fluid jet positive pressure is introducedthrough aperture G and the ear is simultaneously raised (in the Ydimension) and rotated by robot A and motor B, preferably resulting ineach kernel being struck by the fluid jet, causing the embryo andendosperm to be dislodged. The fluid passing through aperture G can beat least one gas, at least one liquid or any combination thereof. Thefluid jet can exert force continuously or non-continuously, for example,as in pulses. As the embryos and endosperms are dislodged by fluid jetpositive pressure from aperture G, they fall down to the shaking screenJ, which retains the endosperms while permitting the embryos to fallthrough to the collecting surface K (for example, sterile cheesecloth)below. Excess fluid can be optionally collected in waste or recyclingreceptacle L. After completion of the embryo removal process for eachcorn ear, the interior of the tube can be briefly washed down manuallyor by automated jet above or below the flange F.

Example 8: Methods of Processing Crude Embryo Preparations

The embryo preparations obtained by methods such as those described inExamples 1 through 7 may include both intact embryos and partialembryos, which may be accompanied by non-embryo tissues, such asendosperm and glumes. Some applications may not require furthertreatment or separation steps, for example, in a mass transformation ofsuch a “crude” embryo preparation where embryos (intact or partial) neednot be separated from non-embryo tissue. For example, callus derivedfrom either intact or partial immature corn embryos can be used fortransformation, regeneration, and production of fertile, transgenicplants. Thus, both intact and partial embryos may serve as transformableexplants and need not be separated from each other. However, in othercases it may be desirable to further purify embryos from a crude embryopreparation.

Procedures wherein some difficulties may be encountered in processingcrude embryo preparations include: (1) rinsing away of non-embryo tissue(e.g., cell debris, starch grains, undesirable proteins), (2)efficiently removing excess liquid from embryos after extrusion orrinsing using liquid, and (3) adding liquid with minimal turbulence sothat the embryos float and do not become submerged.

A porous material is useful for separating non-embryo tissue fromembryos. Any suitable porous material can be employed, preferably havinga mesh or hole size small enough to retain embryos but let smaller,non-embryo tissues or debris pass through, and capable of beingsterilized (e.g., by autoclaving, heat, irradiation, or chemicalsterilization). Suitability of materials is easily judged or tested bysimple experimentation by one skilled in the art. Examples of suitablematerials include cheesecloth or other woven material, and other meshesor screens. In some embodiments, perforated solid materials can be used,including perforated ceramics, polymers, metals, or glasses (forexample, in the form of a Buchner or similar separatory funnel).Cheesecloth of appropriate gauge, for example, has a mesh size smallenough to retain embryos but allows smaller debris to pass through, andis autoclavable. Cheesecloth can be attached to a frame or collar (forexample, the frame holding embryo collecting surface K in FIG. 2 anddescribed in Example 7) to allow the cheesecloth and all the retainedembryos to be simultaneously submerged for easy rinsing. For example,cheesecloth can easily be attached to the frame by means of an elasticband or the like (e.g., silicone tubing); such frames are easilymanufactured, for example, from a beaker or graduated cylinder made ofautoclavable material (e.g., polypropylene, polymethylpentene,polycarbonate, or autoclavable glass) cut into sections. Cheesecloth hasstrong capillarity, allowing liquid to be efficiently pulled away fromthe embryos, thus exposing their waxy epidermis to air prior toflotation. In the flotation step, the cheesecloth is simply submerged inaqueous liquid, allowing the embryos to float off.

Example 9: Substantial Isolation of Embryos Using a Fluid Jet

This example describes a further embodiment of the present invention. Inthis example, multiple embryos were dislodged from seeds by fluid jetpositive pressure.

In the simplest example, a 200-microliter pipette tip was attached to avertical sink nozzle with Parafilm®. When the tap water was turned on ajet emerges from the pipette tip with considerable force. The tap waterpressure was estimated to be about 60 pounds per square inch. This fluid(liquid) jet was trained on an immature corn ear (contained in a beaker)wherein the kernels had been decapitated as described in Example 1. Asthe jet stuck each kernel, the endosperm and embryo were ejected, andcollected in the beaker. Since the endosperm at this stage is arelatively soft tissue it was fragmented into many smaller pieces by thejet, whereas the embryos appeared to remain intact.

The endosperm and embryo tissue dislodged by the jet was poured directlyonto a No. 60 cheesecloth (other suitable porous material, such ashydrophilic mesh of the appropriate mesh size, could be substituted).Different “grades” of cheesecloth are available (for example, grades 10,20, 30, 40, 50, 60, 70, 80, and 90, where the mesh openings decreasewith higher grades), and the grade or mesh size appropriate to theaverage size and shape of a given type of embryo is easily selected bysimple experimentation. The embryos and larger fragments of theendosperm were retained on the upper surface of the cheesecloth. Priorto the next step, the cheesecloth was allowed to partially dry bywicking away excess liquid. This pulled liquid away from the tissues andexposed the surfaces of the embryos to air. When the cheesecloth waslowered into aqueous liquid, the embryos floated because their waxyepidermis did not rewet.

In a simple set up, the cheesecloth (or other suitable porous material)can be manually stretched or held over a receptacle or waste containeras the liquid holding the crude embryo preparations is poured throughthe cheesecloth. For sterile work, the cheesecloth can be attached torigid frames, which can be autoclaved before use. Snap-together sieveswith handles, such as those available in kitchen supply stores, couldalso be used in the method.

Example 10: Devices for Embryo Extraction Using a Fluid Jet

This example describes various embodiments of an apparatus formechanically preparing multiple corn embryos suitable for tissueculture.

One embodiment includes an apparatus for preparing multiple corn embryosusing a fluid jet, generally similar to the device depicted in FIG. 2. Atransparent, open-ended cylinder was made by cutting the ends off a1-liter autoclavable polymethylpentene (PMP) graduated cylinder. Apipette tip (1250-microliter Gilson Distritip, tapered to avoidbackpressure build-up) was secured to the side of the cylinder andserved as an aperture for guiding a fluid stream as a jet through a holemade in the cylinder's wall. Fluid (in this case, water) was fed throughthe pipette tip from PharMed® high pressure autoclavable peristalticpump tubing; the water was delivered from a laboratory sink tap, butcould be an aqueous fluid delivered from a pump or other source. Using apump capable of delivering a sterile fluid is preferable when, forexample, sterile culture medium or a sterile salt solution is found tobe superior to water as a liquid for substantial isolation of embryos.An example of a suitable pump is a Masterflex pump with the highpressure L/S pump head, which can deliver sterile liquid at up to 100psi when used with high pressure tubing.

A corn ear with previously decapitated kernels was manually positionedwithin the cylinder. Once the ear was positioned appropriately withinthe cylinder, each kernel was subjected to positive pressure from thewater jet. This resulted in the embryos and non-embryo tissues beingextruded from the kernels. Examination of the ear after this treatmentindicated efficient removal of the embryos from the kernels. Theextruded material was washed down the cylinder's interior walls to anembryo collector positioned beneath the cylinder. The embryo collectorincluded: (1) a coarse plastic screen (onto which larger debris wastrapped), heat-fused to the cut-off top of a Tri-Pour™ plastic beakerand stacked above (2) a finer screen (Grade 60 cheesecloth, onto whichthe extruded embryos were trapped), secured with an elastic band to thecut-off top of a second Tri-Pour™ plastic beaker and stacked above (3) awaste collection beaker or other container (in which the fine debris,non-embryo tissues, and waste liquid was collected).

Modifications to these and similar embodiments are easily made by oneversed in the art. For example, with regard to positioning the corn earor seed for application of the fluid jet, the ear could be held manuallyin place, or preferably, mounted securely within the cylinder by amovable support capable of moving the ear in three dimensions. Forexample, the ear could be mounted to a threaded metal or polymer rod,such as a polypropylene rod, which could be used to move the ear alongits longitudinal axis as well as to rotate the ear). Another example ofa mounting mechanism is depicted in FIG. 3, which illustrates a magnetic“handle” by which an ear can be secured to a robot arm.

In other embodiments, however, the corn ear or ears need not beindividually secured to a holder but can be freely movable so as toallow multiple kernels to be contacted by the force used to remove theembryos from the kernels. For example, at least one ear, or multipleears, can be borne on or held between at least one support, such as, butnot limited to, at least one plane, frame, grid, screen, mesh, platform,roller, guide wire or rod, and belt, wheel, or roller conveyor. Such asupport could be movable or could cause the ear or ears to move, forexample, by vibration, rolling motion, gravity, or other mechanisms.Substantially isolated embryos could pass through the platform itself ifthe platform was porous (e.g., made of mesh). The ear or ear can also befloated on a fluid in a manner allowing each ear to rotate or otherwisemove freely while afloat. The fluid, such as a liquid containing thesubstantially isolated embryos, could be continually drained off,optionally through a filtering or sedimenting device, or collected forcentrifugation.

Devices for obtaining motion along the longitudinal axis of a corn earinclude, but are not limited to, ball screw-driven slides or belt-drivenslides, such as those commercially available from various manufacturerssuch as Techno, Inc. (techno-isel.com). To obtain rotary motion forrotating a corn ear, a stepper motor can be used, for example, a steppermotor attached to a slide plate. Rotary motion can also be provided byrolling devices, for example, by parallel round or tubular rollersbetween which the corn ear is held and rotated.

The shape of the fluid jet can be advantageously modified according tothe desired application. For example, a narrow column-shaped jet ofuniform diameter is useful for removal of embryos from one seed at atime. Where it is desirable to increase the rate at which embryos aresubstantially isolated, multiple embryos can be simultaneously removedfrom their seed by a fluid jet; this can be achieved, for example, byusing at least one single fluid jet that covers a larger area, or byusing multiple jets simultaneously. In one embodiment, multiple jets,such as multiple parallel, narrow, column-shaped jets (for example,produced by multiple nozzles similar to that used in Example 9 andoptionally connected to each other by a manifold) are used to directfluid jet positive pressure on multiple seeds to substantially isolatetheir embryos substantially simultaneously. Automation of these andother devices can further include optical or mass sensors to aid inpositioning the ear and fluid jet relative to each other.

In another embodiment, at least one fluid jet that covers a larger area(for example, wherein the fluid jet simultaneously impacts multiplekernels, or multiple rows of kernels on a corn ear) can be used. Thedimensions of such a jet preferably allow the jet to enter the kernelsand wash out the embryo. Typically, corn embryos used in geneticexperiments are immature and generally in the size range of about 1.8 toabout 2.2 millimeters in length; the kernels holding these immatureembryos are generally in the size range of between about 4 and about 5millimeters in width. For embryos of this size, an appropriate fluid jetcan be, for example, between about 0.5 to about 1 millimeter in width.

Any suitable means for producing such a larger fluid jet may be used,such as, but not limited to nozzles that generate non-columnar fluidjets. Examples of suitable nozzles include, but are not limited to,nozzles that generate a flat spray pattern and nozzles that generate afan- or a cone-shaped spray pattern. In one example, a commerciallyavailable flat spray nozzle (number 23990-1/4-04, Spraying Systems Co.,Dillburg, Pa.) was used with a Masterflex L/S pump (model 77250-62) topump liquid at 1 liter per minute and 30 psi; embryos were excised froma corn ear under these conditions. Another example of a preferred nozzleis a nozzle that generates a fluid jet in the form of a flat “sheet” offluid, such as is depicted in FIG. 4. Such a nozzle preferably iscapable of generating a uniform, flat fluid jet that maintains acoherent, uniform sheet-like flow for at least a distance sufficient toallow the flow to contact more than one seed (and preferably severalseeds) at the same time. The novel nozzle depicted in FIG. 4 is designedto generate a uniform, flat sheet-like jet that is about 0.5 to about 1millimeter in thickness, greater than about 20 millimeters in width, andmaintains the sheet-like flow over a distance of about 20 to about 25millimeters from the nozzle's aperture. This latter distance permits thejet to be moved along the rows of kernels with minimal adjustment neededfor differences in distance between the surface of the kernels and thenozzle's aperture.

Regardless of the area or shape of the jet or spray pattern generated bythe nozzle or aperture through which the liquid flows, nozzles orapertures are preferably used with flow rates and pressures sufficientto generate enough fluid force to dislodge the embryo from its seed,without damage to the embryo. In some embodiments, it is preferable touse a lower flow rate and possibly a higher pressure, to minimizeconsumption of fluid (such as medium) as well as to minimize the wastegenerated.

Example 11: Using a Gas Jet to Substantially Isolate Embryos

This example describes further embodiments of methods and devices formechanically preparing multiple corn embryos suitable for genetictransformation or tissue culture. As described in Example 6, gas jetscan also be used for the substantial isolation of multiple embryos. Anapparatus similar to that described in Example 10 was modified for usewith gas. A 1-milliliter pipette tip (catalogue number TN1300RS, MarshBio Products) was secured to the side of the cylinder and served as anaperture for guiding a stream of air as a jet through a hole made in thecylinder's wall. Air was supplied from a compressor pressurized tobetween about 60 to about 100 psi. An air valve for convenience waspositioned in line between the compressor and the pipette tip. The airjet emerging from this pipette tip was used to dislodge the embryos froma prepared corn ear. Examination of the kernels after they had beensubjected to the air jet showed that the thick pericarp remained inplace and surrounded by papery glumes, and the pericarp contents (embryoand endosperm) had been removed. Examination of the tissue retained bythe grade 60 cheesecloth showed that this included dislodged embryos aswell as some glumes dislodged by the high-pressure air jet. The glumesof corn have a waxy surface like the embryos and also float followingthe flotation procedure. Using lower air pressures can reduce glumecontamination.

Example 12: Substantial Isolation of Embryos Using Other Fluid Forces

This example describes further embodiments of methods and devices formechanically preparing multiple corn embryos suitable for genetictransformation or tissue culture. Forces exerted by fluids, other thanpositive fluid pressure from a fluid jet, can be used to substantiallyisolate embryos. In one experiment, the tops of kernels were removedfrom a corn ear, which was placed inside a bottle containing steriledistilled water and shaken vigorously by hand. This resulted in thesubstantial isolation of 90 out of the ear's 200 embryos. Anotherexperiment repeated the preceding procedure except that the shaking wascarried out in a mechanical paint shaker. In this experiment, 56 embryoswere substantially isolated out of the ear's 190 embryos. In a thirdexperiment, a similar procedure was carried out, except that the cornear was pre-soaked in 211 medium, and the shaking was carried out in apaint shaker. In this experiment, 109 embryos were substantiallyisolated out of the ear's 210 embryos. In these cases, non-jet fluidforce from movement of the liquid around the corn ear resulted in thesubstantial isolatation of the embryos; the fluid force could includefluid turbulent flow, fluid laminar flow, shear from fluid flow,negative fluid pressure (for example, resulting cavitation), orcombinations thereof. Forces can also include forces generated byacoustic techniques, such as by an acoustic wave or waves (pulsed orcontinuous) in either gas or fluid phase.

Preceding examples (including Examples 9-11) described use of a fluidjet to remove embryos from an immature ear. During these procedures, itwas observed that the fluid jet generally also caused at least part ofthe endosperm to be released from the kernel. The endosperm tissue wasobserved to be softer and more friable than the embryos, and tended todisintegrate to varying degrees (in contrast to the embryos, whichtended to remain intact). It is possible that the endospermsdisintegrate upon exposure to shear caused by the fluid jet. This shearis believed to be non-uniform, resulting in the variability indisintegration observed; nonetheless, a large proportion of theendosperm material that was sufficiently disintegrated to pass throughthe cheesecloth, leaving a retentate made up of a semi-pure preparationof embryos.

When a low-pressure jet from an ordinary laboratory squirt bottle wasdirected at the cheesecloth retentate, more of the remaining endospermtissue was disintegrated further and washed through the cheesecloth,leaving behind a relatively more pure preparation of embryos. Thus it isreasonable to predict that if the retentate is uniformly exposed to ashear force of the correct intensity, all or substantially all of theremaining endosperm should disintegrate and pass through thecheesecloth. Such a shear force could be generated by any suitablemeans, such as, but not limited to, a single jet, multiple jets, asheet-like or curtain-like jet, rapidly moving jets, and acceleration ordeceleration of the endosperms. Additionally, if the jet used toinitially release the kernel contents is designed to expose a higherproportion of the endosperms to shear during ejection, an initial higherpurity embryo preparation could be obtained.

One non-limited embodiment of applying shear to further purify embryosfollows. Once the embryos and partially disintegrated endosperms arereleased from a cob, the remainder of the endosperm can be rapidlyfragmented by fluid flow, for example, from a spray nozzle, that strikesthe endosperm uniformly and simultaneously. One suitable type of nozzleis a full cone nozzle. Full cone nozzles generate a spray patterncompletely filled with drops. An internal vane within the nozzle impartscontrolled turbulence to the liquid prior exiting to the orifice,allowing formation of the spray pattern. Commercially available nozzleshave spray patterns that are round, square, or oval. An example of asuitable full cone nozzle is known as “UniJet Spray Nozzle, StandardSpray, Small Capacity” (part number TG-SS0.3, MANUFACTURER/LOCATION?).

Example 13: Combination Devices

This example describes several additional embodiments of the method ofthe invention, which use a combination of forces to substantiallyisolate multiple embryos from seeds.

FIG. 5 illustrates a device using a larger fluid jet (as described inExample 10). This device includes a nozzle for generating a fluid flowsuch as a larger fluid jet (for example a flat fluid jet), and,optionally, a suction head, or component for applying negative fluidpressure (e.g., by vacuum or suction), for dislodging embryos and/or forcollecting the dislodged embryos. FIG. 5 (top) depicts a cross-sectionalview of an example of such a device, showing how the nozzle, optionalsuction head, and corn ear can be positioned relative to each other. Thecorn ear, nozzle, and optional suction head can be moved relative toeach other; for example, the corn ear may be stationary while the nozzleand optional suction head are moved, or the nozzle and suction head maybe stationary while the corn ear is moved. FIG. 5 (bottom) schematicallydepicts a corn ear positioned in the device, and shows the nozzlepositioned to generate a flat fluid jet wherein the jet impacts multiplekernels in a row.

FIG. 6 depicts an embodiment of a suitable suction head or component forapplying negative fluid pressure (e.g., by vacuum or suction), such asis optionally used in the device of FIG. 5, and which can also be usedon its own to substantially isolate embryos. The suction head caninclude one or more apertures through which negative fluid pressure canbe applied. The suction head can also include a means for dispensingfluid (such as gas or liquid, e.g., water or medium), for example,multiple apertures in the suction head. For use with corn, the suctionhead is preferably shaped to follow the contours of a typical corn ear,and is preferably capable of entrapping embryos from multiple kernels orfrom multiple rows of kernels. It is envisioned that the suction headcan be manufactured of a rigid material (such as stainless steel orother metals), or of a flexible material to allow easier conformation ofthe suction head to the contours of a corn ear, or of combinationsthereof. Embryos can be substantially isolated by any combination ofmechanical positive pressure (exerted, for example, by a leading edge ofthe suction head), negative fluid pressure (e.g., suction or vacuum),and fluid force (such as, but not limited to, positive pressure from afluid jet, fluid turbulent flow, and fluid laminar flow entrappingmaterial from the interior of the kernel)

Devices for applying force for substantially isolating embryos, such asare described in Examples 1, 3, 4, 6, 7, 9, 10, and the present example(including, but not limited to the devices illustrated in FIGS. 5 and 6)can be moved relative to the corn ear. The ear may be stationary, or thedevice may be stationary, or both can be moved. Because corn seedtypically occurs in relatively uniform rows arranged parallel to thelongitudinal axis of the corn ear, the device is typically moved(relative to the ear) so that the device passes parallel to thelongitudinal axis of the corn ear and following a row or multiple rowsof kernels. However the motion of such devices relative to the ear canfollow the circumference of the ear, or can be random, or can be anycombination of suitable motions.

FIG. 7A through 7C depict different views of an embodiment of a devicethat uses a combination of forces to substantially isolate multipleembryos from seed (in this example, corn). This device includes a headwith a leading edge capable of applying a predefined amount ofmechanical pressure to the base of kernels that previously have had thepericarp opened or truncated, so that the embryos are extruded from thekernels in a manner similar to those described in Examples 1, 3, and 4.The device further includes a component for applying negative fluidpressure (e.g., by vacuum or suction) for dislodging embryos and/or forcollecting the dislodged embryos. The extruded embryos (and accompanyingnon-embryo tissues) are thus separated from the corn ear and can becollected by application of negative fluid pressure. The collectedembryos and non-embryo tissues can be further separated, if desired, bysuitable means, such as by size-separation, hydrophobic separation, ordifferential centrifugation. A variation of this device could include ameans for dispensing fluid (such as liquid, e.g., water or medium), forexample, multiple apertures in the suction head.

The embryo extraction devices depicted in FIGS. 5, 6, and 7 aredescribed as illustrative examples that are not intended to be limiting.These and other such devices can include additional components, forexample, means for separating the embryos from non-embryo tissues orfrom fluids used in the substantially isolation process.

Example 13: Viability Data

The multiple monocot embryos provided by use of the methods and devicesof the present invention are most preferably embryos suitable forgenetic transformation or tissue culture application such astransformation and regeneration of plants. This example furtherillustrates the utility of methods of the invention in providingmultiple monocot embryos that are viable and suitable for genetictransformation or tissue culture. In this example, the quality ofimmature corn embryos obtained by different excision methods wascompared in their response to transformation by Agrobacteriumtumefaciens.

For transformation, a plasmid containing left and right bordersequences, a gene for glyphosate resistance for selection, and areporter gene (gfp, encoding green fluorescent protein) was used.Agrobacterium containing this plasmid was streaked from a frozenglycerol stock onto an LB plate and grown for 3 days in a 28 degreeCelsius incubator. A seed culture was prepared by inoculating threecolonies from the LB plate into 25 milliliters of LB broth, which wasincubated 15 hours at 27 degrees Celsius with shaking (200 rpm). Thisseed culture (10 milliliters) was diluted with 40 milliliters of freshLB broth and grown for 6 hours at 200 rpm at 27 degrees. Agrobacteriumwas centrifuged for 10 minutes, and the pellet resuspended at an opticaldensity of 0.2 at 660 nanometers in AB minimal induction media. This wasincubated 15 hours at 27 degrees Celsius with shaking (200 rpm). TheAgrobacterium culture was centrifuged for 10 minutes and the pelletwashed with 10 milliliters Lynx 1040 and resuspended in 5 millilitersinoculation medium. The optical density was adjusted to 1.0 and used forinoculation.

Four experiments (designated A, B, C, and D respectively) wereperformed. Each experiment compared embryos obtained by manual excisionto embryos obtained by a method of the present invention: excision by aliquid jet (experiments A, B, and D) or excision by a gas jet(experiment C). The liquid jet in experiments A and B used ordinary tapwater and a nozzle made of a pipette tip. Experiment C tested a gas jetusing air from a compressed-air pump and a nozzle made of a pipette tip.The liquid jet in experiment D used ½ MSPL medium as the liquid and asolid stream nozzle with an equivalent orifice diameter of 0.020 inches(catalogue number TP000050-SS, Agricultural Division of Spraying SystemsCo., Dillburg, Pa.).

Corn ears were harvested twelve days after pollination and sterilized bysoaking in a 1-liter bottle of 80% ethanol for 3 minutes. Embryos weremanually excised by cutting off the top third of the kernel with ascalpel and removing the embryo from the kernel using a narrow spatula.The collected embryos were excised into 1 milliliter of ½ MSPL medium ina single microcentrifuge (Eppendorf) tube. The medium was removed andreplaced with 1 milliliter of Agrobacterium tumefaciens prepared asdescribed below

Embryos were also substantially isolated using a fluid (liquid or gas)jet, following procedures similar to those described in Examples 10 and11. The fluid jet was used to excise the remaining embryos on the earsafter removing the top third of the kernel with a scalpel. The ear waspositioned so that the fluid jet was aimed into individual cut kernelsin succession to dislodge both the embryo and non-embryo tissue(endosperm). The kernel contents removed from the ear were passedthrough a coarse screen to remove large pieces of endosperm, and theembryos were collected on sterile cheesecloth. Embryos were transferredusing a small spatula from the cheesecloth into a microcentrifuge tubecontaining 1 milliliter of ½ MSPL medium. After all of the embryos werecollected, ½ MSPL medium was removed and replaced by 1 milliliter ofAgrobacterium tumefaciens inoculant.

Embryos prepared by the various excision methods were subjected to thesame inoculation, selection, and regeneration procedures. Suitableprocedures, including descriptions of media and reagents, fortransformation of plants using glyphosate selection and GFP as areporter have been disclosed in United State Patent ApplicationPublication Number 2004/0244075 to Cai et al., which is incorporated byreference in its entirety herein.

Embryos were inoculated with 1.0 milliliters of Agrobacterium for 5minutes. The contents of the microcentrifuge tube were poured onto aplate of co-culture medium, and co-cultured for 18 hours at 23 degreesCelsius. Embryos were transferred next to induction MS medium, andcultured at 30 degrees Celsius for 13 days. Calli derived from thetransformation were cultured at 27 degrees Celsius for 11 days prior toregeneration. At this time, GFP positive sectors were counted using afluorescence microscope. For regeneration, calli derived from eachembryo were individually transferred to MS/6 BA medium and cultured in alight room for 7 days, after which each greening callus was transferredto MSOD medium and returned to the light room for 17 additional days.Resulting shoots were transferred to Phytatrays containing regenerationmedium (consisting of 2.165 g MS basal salts, 5 milliliters 100×MSvitamins, and 20 grams sucrose made up to 1 liter in water andautoclaved, pH adjusted with KOH to 5.8, solidified by autoclaving with3 g Phytagel, and with 0.75 milliliters of 1 milligram per milliliterindole-3-butyric acid, 0.5 milliliters of 1 milligram per milliliter1-naphthaleneacetic acid, and 0.2 milliliters 0.5 molar glyphosateadded). After about 3 weeks, transgenic plants were hardened off bytransplanting rooted shoots in peat pots containing soil mix and grownat 26 degrees Celsius.

The results of these experiments are summarized in Table 1. The numberof embryos that were transformable is estimated from the number ofGFP-positive embryos. Overall transformation and regeneration frequencyis given as the percentage of GFP-positive plants regenerated from theinoculated embryos. These results demonstrate that various methods anddevices of the present invention are useful for providing multiplemonocot embryos suitable for genetic transformation or tissue culture.

TABLE 1 number number trans- ex- number of of GFP- trans- of formation/peri- excision embryos positive formation plants to regeneration mentmethod inoculated embryos frequency soil frequency A manual 56 23 41% 6 11% liquid jet 44 8 18% 3 6.8% B manual 22 11 50% 6  27% liquid jet 234 17% 1   4% C manual 33 27 82% n/a n/a gas jet 61 19 31% n/a n/a Dmanual 36 17 47% n/a n/a liquid jet 166 51 31% n/a n/a n/a: data notavailable

All of the materials and methods disclosed and claimed herein can bemade and used, as instructed by the above disclosure, and without undueexperimentation, by a person of ordinary skill in the art. Although thematerials and methods of this invention have been described in terms ofpreferred embodiments and illustrative examples, it will be apparent tothose of skill in the art that variations may be applied to thematerials and methods described herein without departing from theconcept, spirit, and scope of the invention. All such similarsubstitutes and modifications apparent to those skilled in the art aredeemed to be within the concept, spirit, and scope of the invention asfurther defined by the appended claims.

1. A method of providing monocot embryos suitable for transformation ortissue culture, comprising: (a) providing monocot seeds containingimmature embryos having an opening in the pericarp of said seeds; and(b) applying force to said seeds sufficient to extract said immatureembryos from said seeds, wherein said extracted immature embryoscomprise embryos or pieces of embryos suitable for genetictransformation or tissue culture wherein the force is selected from thegroup consisting of: negative pressure, centrifugal force, linearacceleration, linear deceleration, bulk maceration, vibration, andultrasound.
 2. The method of claim 1, further comprising separating saidextracted immature embryos from associated non-embryo tissue, whereinsaid separated extracted immature embryos comprise embryos suitable fortissue culture.
 3. The method of claim 1, wherein said monocot is in thefamily Poaceae.
 4. The method of claim 1, wherein said monocot is a Zeaspecies.
 5. The method of claim 4, wherein said multiple monocot seedscomprise multiple corn kernels on at least one corn ear.
 6. The methodof claim 1, wherein said embryos suitable for tissue culture compriseintact embryos.
 7. The method of claim 1, wherein said embryos suitablefor tissue culture comprise partial embryos.
 8. The method of claim 1,wherein said tissue culture comprises transformation.
 9. The method ofclaim 1, wherein said tissue culture comprises regeneration.
 10. Themethod of claim 9, wherein said regeneration results in callusformation.
 11. The method of claim 9, wherein said regeneration resultsin at least one fertile plant. 12-13. (canceled)
 14. The method of claim2, wherein said separating comprises use of at least one ofsize-exclusion, hydrophobic separation, and density differentials.15-27. (canceled)
 28. The method of claim 5, wherein the corn ear isimmature.
 29. The method of claim 1, wherein the pericarp is opened byusing one or more of the following: a needle, an awl, a blade, anabrasive material, application of a chemical treatment, and applicationof an enzymatic treatment.